The industrial production of the amino acid lysine has became an economically important industrial process. Lysine is used commercially as an animal feed supplement, because of its ability to improve the quality of feed by increasing the absorption of other amino acids, in human medicine, particularly as ingredients of infusion solutions, and in the pharmaceutical industry.
Commercial production of this lysine is principally done utilizing the gram positive Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum (Kleemann, A., et. al., “Amino Acids,” in ULLMANN'S ENCYCLOPEDIA OF INDUSTRIAL CHEMISTRY, vol. A2, pp. 57-97, Weinham: VCH-Verlagsgesellschaft (1985)). These organisms presently account for the approximately 250,000 tons of lysine produced annually. A significant amount of research has gone into isolating mutant bacterial strains which produce larger amounts of lysine. Microorganisms employed in microbial process for amino acid production are divided into 4 classes: wild-type strain, auxotrophic mutant, regulatory mutant and auxotrophic regulatory mutant (K. Nakayama et al., in Nutritional Improvement of Food and Feed Proteins, M. Friedman, ed., (1978), pp. 649-661). Mutants of Corynebacterium and related organisms enable inexpensive production of amino acids from cheap carbon sources, e.g., molasses, acetic acid and ethanol, by direct fermentation. In addition, the stereospecificity of the amino acids produced by fermentation (the L isomer) makes the process advantageous compared with synthetic processes.
Another method in improving the efficiency of the commercial production of lysine is by investigating the correlation between lysine production and metabolic flux through the pentose phosphate pathway. Given the economic importance of lysine production by the fermentive process, the biochemical pathway for lysine synthesis has been intensively investigated, ostensibly for the purpose of increasing the total amount of lysine produced and decreasing production costs (reviewed by Sahm et al, (1996) Ann. N.Y. Acad. Sci. 782:25-39). There has been some success in using metabolic engineering to direct the flux of glucose derived carbons toward aromatic amino acid formation (Flores, N. et al., (1996) Nature Biotechnol. 14:620-623). Upon cellular absorption, glucose is phosphorylated with consumption of phosphoenolpyruvate (phosphotransferase system) (Malin & Bourd, (1991) Journal of Applied Bacteriology 71, 517-523) and is then available to the cell as glucose-6-phosphate. Sucrose is converted into fructose and glucose-6-phosphate by a phosphotransferase system (Shio et ah, (1990) Agricultural and Biological Chemistry 54, 1513-1519) and invertase reaction (Yamamoto et al, (1986) Journal of Fermentation Technology 64, 285-291).
During glucose catabolism, the enzymes glucose-6-phosphate dehydrogenase (EC 1.1.14.9) and glucose-6-phosphate isomerase (EC 5.3.1.9) compete for the substrate glucose-6-phosphate. The enzyme glucose-6-phosphate isomerase catalyses the first reaction step of the Embden-Meyerhof-Parnas pathway, or glycolysis, namely conversion into fructose-6-phosphate. The enzyme glucose-6-phosphate dehydrogenase catalyses the first reaction step of the oxidative portion of the pentose phosphate cycle, namely conversion into 6-phosphogluconolactone.
In the oxidative portion of the pentose phosphate cycle, glucose-6-phosphate is converted into ribulose-5-phosphate, so producing reduction equivalents in the form of NADPH. As the pentose phosphate cycle proceeds further, pentose phosphates, hexose phosphates and triose phosphates are interconverted. Pentose phosphates, such as for example 5-phosphoribosyl-1-pyrophosphate are required, for example, in nucleotide biosynthesis. 5-Phosphoribosyl-1-pyrophosphate is moreover a precursor for aromatic amino acids and the amino acid L-histidine. NADPH acts as a reduction equivalent in numerous anabolic biosyntheses. Four molecules of NADPH are thus consumed for the biosynthesis of one molecule of lysine from oxalacetic acid. Thus, carbon flux towards oxaloacetate (OAA) remains constant regardless of system perturbations (J. Vallino et al, (1993) Biotechnol. Bioeng., 41, 633-646).